Standard pathological determinations of tissue biopsies rely almost exclusively on the visual examination of tissue and cellular morphology. As successful as these conventional analyses are in determining the presence and extent of a carcinoma, they are largely subjective, and there is a chance that errors can be made in the diagnosis of the presence or progression of the disease. The search for more quantifiable methods of disease determination has significant appeal. Ideally, a method that maintains existing visualization protocols, while simultaneously adding a quantitative analytical aspect, would be extremely powerful. In a continuing series of studies, we are investigating the application of newly developed vibrational spectroscopic imaging microscopies, as a tool for quantitatively identifying the presence and progression of the diseased state. These techniques integrate microscopy with vibrational spectroscopy, yielding images that exhibit contrast based on intrinsic biochemical variations within tissue sections. These methods maintain the visualization capabilities essential for histological protocols, while simultaneously building on the extensive capabilities of conventional single point infrared and Raman spectroscopy. The ultimate goal of our investigations is to examine whether infrared and Raman spectroscopic imaging microscopy are viable and sensitive approaches for tracking the progression of tumorigenesis. An additional advantage of these techniques is that chemical data on thousands of individual cells in a tissue section are recorded in a single imaging experiment. This enables the application of a variety of multivariate and statistical methods to analyze the data and to potentially chart the progression and outcome of the disease. To date we have used infrared and Raman spectroscopic imaging to visualize spatial variations in bulk chemical composition in a variety of tissue types at the cellular level. Specifically, we have monitored disease induced changes in lipid and protein distributions as well as cellular DNA, RNA and glycogen levels. In addition, we are able to image spatial variations in cell membrane structure and protein secondary structure within individual tissue sections. Promising preliminary results with ductal carcinoma and normal human breast cell lines were the impetus for extending the work to murine breast tumor tissue and human prostate tissue. Studies are presently underway to perform infrared and Raman spectroscopic imaging of these tissues and to build an imaging spectral library from a variety of normal and diseased tissue types. These data will enable us to refine our statistical and analytical protocols. It is anticipated that this effort will ultimately provide a strong foundation for visualizing the presence and quantifying the extent of disease.